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    The Role of Estrogen Receptor Coactivators in Tamoxifen Resistance

    Scientific Abstract:
    The Role of Estrogen Receptor Coactivators in Tamoxifen Resistance. Background: Breast cancer is one of the most widespread malignancies among women in the western world and the role of estrogen (E) in contributing to and propagating breast tumor growth is widely accepted. Generally, E exerts its effects by signaling to the estrogen receptor (ER) of which there are two subtypes, ER-alpha and ER-beta. When E binds to ER, ER changes conformation, dimerizes, and binds to estrogen response elements (ERE) upstream of the E-target gene. The ER complex then recruits coactivators that are involved in enhancing ER-mediated gene transcription and the expression of target genes. The most studied steroid receptor coactivators (SRCs) associated with the ER are SRC-1, -2 (TIF2) and -3 (RAC3). SRC-3, in particular, is amplified in breast cancer-1 (AIB1) and further evidence suggests that the levels of these coactivators determine tamoxifen’s (TAM’s) ability to function as either an agonist or antagonist in target tissue. In the breast, TAM is an antiestrogen and thus blocks E-mediated signaling to the ER. TAM also recruits corepressors to the ER complex instead of coactivators, which prevents gene transcription and expression of E-regulated genes. Unfortunately, in many patients, TAM therapy becomes ineffective because the cancer develops into an E-independent and TAM-resistant phenotype. Objective/Hypothesis: We predict that coactivator levels in breast tissue play a role in TAM resistance during breast cancer. This is likely since ER-mediated signaling is involved in breast cancer tumorigenesis and SRC family coactivators are involved in enhancing ER-mediated gene transcription. Thus, the main objective of this research is to determine the role of SRC family coactivators in E-dependent, E-independent and TAM-resistant breast cancer phenotypes. We propose that differences in the following parameters are likely between E-dependent, E-independent and TAM-resistant breast cancer: a) cell proliferation b) activation of ER-responsive genes c) cell cycle kinetics and d) apoptosis. Thus, we hypothesize that depletion of SRC family coactivators will restore the TAM-sensitive phenotype of TAM-resistant cells by altering cell cycle kinetics and inducing apoptosis. We propose the following specific aims to test our hypothesis. Specific Aims: (1) Determine the role of SRC family coactivators in E-dependent, E-independent and TAM-resistant breast cancer cell growth. (2) Assess if SRC family coactivators affect the activity of ER-regulated genes in E-dependent, E-independent and TAM-resistant breast cancer. (3) Examine the effect of SRC family coactivator depletion on cell cycle and apoptotic factors as mechanisms for E independence and TAM resistance during breast cancer. Study Design: In these studies we will use three mammary cell lines that serve as an in vitro model for breast cancer progression. The parental MCF-7 cell line depends on E for growth and is sensitive to treatment with TAM. In contrast, the LCC1 and LCC2 cell lines were derived from MCF-7 cells in ovariectomized nude mice and therefore do not require E for growth. In addition, the LCC2 cell line was further differentiated from the LCC1 cell line in the presence of high TAM until it evolved into a TAM-resistant cell line. We will employ antisense oligonucleotide (asODN) and small inhibitory RNA (siRNA) technologies to knockdown gene expression of SRC-1, TIF-2 and RAC-3 to determine the role of coactivators on cell proliferation in the context of agonist or antagonist treatment. If changes in proliferation are observed, as we anticipate, we will also evaluate cell cycle parameters and apoptotic factors as potential mechanisms to explain the development of E-independent and TAM-resistant phenotypes during breast cancer. Potential Outcomes and Benefits of the Research: TAM is commonly used for the treatment of breast cancer but through the course of treatment many women acquire a resistance to therapy. This study aims to understand the role that the SRC family coactivators associated with the ER play in TAM resistance. This is important clinically because many women succumb to breast cancer as a result of inefficient treatment. The proposed project is also important from a public health perspective because it is necessary for women to understand how E and TAM work in their body in order to make more informed decisions about their health and subsequent medical treatment. We anticipate that this study will lead to a better understanding of E independence and TAM resistance during breast cancer with the long-term goal of developing more successful treatments for the disease.

    Lay Abstract:
    The Role of Estrogen Receptor Coactivators in Tamoxifen Resistance. Next to skin cancer, breast cancer is the most common cancer among women in the United States. The American Cancer Society estimates that in 2003, approximately 211,000 women will find out they have breast cancer. Gravely, it is further predicted that 40,000 women will die from breast cancer during this same time. Generally, estrogen (E) promotes the typical growth and development of breast tissue. Unfortunately, life exposure to E also increases a woman’s chances of being diagnosed with breast cancer. During breast cancer, E stimulates the breast tumor to grow by signaling to the estrogen receptor (ER) of which there are two kinds, ER-alpha and ER-beta. Once E binds to ER, ER alters it’s shape, comes together with another ER and binds to estrogen response elements (ERE) in the DNA located upstream of the E-target gene. These changes allow the ER unit to recruit coactivators, which amplify ER-mediated gene transcription and the expression of target genes. The most studied steroid receptor coactivators (SRCs) associated with the ER are SRC-1, -2 (TIF2) and -3 (RAC3). SRC-3 is amplified in breast cancer (AIB1) and further evidence suggests that the levels of SRC family coactivators are likely to influence tamoxifen’s (TAM’s) ability to function as either an agonist or antagonist in target tissue. Due to TAM’s tissue-specific ability to be either an agonist or an antagonist, it is considered a selective estrogen receptor modulator (SERM). In the breast, TAM is an antagonist and therefore blocks E-mediated signaling to the ER. TAM also recruits corepressors to the ER complex instead of coactivators and thus prevents gene transcription and expression of E-regulated genes. Initially, TAM is an effective treatment for breast cancer because the tumor is E-dependent and TAM-sensitive. In its more malignant stages, the breast tumor can grow in the absence of E and further develops a TAM-resistant phenotype. Unfortunately, the reasons for the evolution of this phenotype are not fully understood. Although there are numerous explanations postulated, we predict that the levels of SRC family coactivators present in the breast tissue are likely to play a key role in the E-independent and TAM-resistant phenotypes of breast cancer. Thus, the main goal of this research is to determine the role that SRC family coactivators play in the growth of E-dependent, E-independent and TAM-resistant breast cancer cells. We propose to study this in three mammary cell lines (MCF-7, LCC1 and LCC2) that will serve as an in vitro model for breast cancer. The parental MCF-7 cell line depends on E for growth and is sensitive to treatment with TAM. In contrast, the LCC1 and LCC2 cell lines were derived from MCF-7 cells in ovariectomized nude mice and therefore do not require E for growth. In addition, the LCC2 cell line was further differentiated from the LCC1 cell line in the presence of increasing levels of TAM until it changed into a TAM-resistant cell line. In these cell lines, we will use gene-silencing technologies to reduce levels of SRC-1, TIF2 and RAC3. We will then evaluate the following factors: a) cell proliferation b) activation of ER-responsive genes c) cell cycle kinetics and d) apoptosis. This will allow us to assess if reducing certain coactivators affects the growth of E-dependent, E-independent and TAM-resistant breast cancer cells in response to treatment with E or TAM. Furthermore, we can determine which ER-regulated genes are affected by reduced coactivator levels and if changes in cell cycle and apoptotic factors among the cells can explain differing endocrine sensitivities during breast cancer. Overall, this study aims to understand the role that SRC family coactivators play in TAM resistance. We anticipate that the role of coactivators as common mediators in both E and TAM-mediated signaling through the ER will be better understood. This can benefit breast cancer patients who fear the recurrence of their cancer due to TAM resistance by developing therapies that don’t depend on ligand-based anti-estrogen treatment. Thus, the idea is to bypass TAM resistance by providing alternative treatments that take into consideration TAM-mediated action through the ER. We anticipate that this study will lead to a better understanding of E independence and TAM resistance during breast cancer with the long-term goal of developing more successful treatments for the disease.